A baculovirus expression system enables high-level expression of a gene of interest by causing recombination of the gene in Sf9 cells using a promoter of a polyhedrin gene of baculovirus. A polyhedrin is expressed in the nucleus of Sf9 cell at a high level as an occlusion body which is a form to be used when viruses become latent within the cell. The baculovirus expression system where a recombinant protein is introduced into a polyhedrin gene and the expressed proteins are purified, has many advantages over that of Escherichia coli or yeast, such that the expressed proteins having a lot of hydrophobic regions such as membrane-bound proteins are comparatively hard to agglutinate and the expressed proteins undergo a posttranscriptional modification which is necessary for protein functions, such as addition of sugar chains and coordination of metal ion, and therefore the baculovirus expression system is often used as an expression system of membrane receptor protein (Tate C G, Grisshammer R., Trends in Biotechnology 1996, 14, pp 426-430, Heterologous expression of G protein-coupled receptors; and, Grisshammer R, Tate C G, Quarterly Reviews of Biophysics 1995, 28, pp 315-422, Overexpression of integral membrane proteins for structural studies).
Baculovirus has another life cycle in addition to that a baculovirus becomes a polyhedrin virus coated with polyhedrin and present in the nucleus. In order to proliferate and infect, baculovirus becomes a budded virus (Budded virus: this is also referred to as budded baculovirus in this specification), rupturing Sf9 cell membrane and being released outside the cell. Loisel et al have reported that at this time a receptor of seven-transmembrane type recombined into the above polyhedrin protein is expressed on the cell membrane and recovered from the envelope of the budded baculovirus (Loisel T P, Ansanay H, St-Onge S, Gay B, Boulanger P, Strosberg A D, Marullo S, Bouvier M., Nat Biotechnol. 1997 November; 15(12): 1300-4., Recovery of homogeneous and functional beta 2-adrenergic receptors from extracellular baculovirus particles). It has also been reported that, whereas most receptors of seven-transmembrane type expressed in a host cell have a sugar chain structure which is not functional, only functional receptors are recovered from the viral envelope.
It has also been reported that a group of proteins which are present in endoplasmic reticulum (ER) membrane or Golgi apparatus membrane and are involved in the intracellular cholesterol feedback regulation, such as SREBP (sterol regulatory element binding protein)2, HMG-CoA (hydroxymethyl glutaryl coenzyme A) reductase, SCAP (SREBP cleavage activating protein), S1P (site 1 protease), are expressed on the virus envelope with maintaining their functions (Ishihara, G, Shirai, H., Yamaguchi, M., Fukuda, R., Hamakubo, T., and Kodama, T., Atherosclerosis, 151, p 290, 2000, Expression of cholesterol regulatory proteins on extracellular baculoviruses).
On the other hand, G protein coupled receptors (GPCRs) are important as a target for development of medicament, and about 700 receptors are reported in the genome database (Venter J G, Adams M D, Myers E W, et al., Science 291, pp 1304-1351, 2001, The sequence of the human genome). The mechanism of the signal transduction of hormone has also been studied (Tate C G, Grisshammer R., Trends in Biotechnology 1996, 14, pp 426-430, Heterologous expression of G protein-coupled receptors). GPCR has seven transmembrane domains, and is coupled with heterotrimeric G protein. The type of α subunit of G protein which is bond (coupled) when binding with ligand is determined depending on the type of receptor. For example, in the case of the leukotriene B4 receptor, it is Gi or Gq class G protein (Igarashi T, Yokomizo T, Tsutsumi O, Taketani Y, Shimizu T and Izumi T., Eur. J. Biochem., 259, pp 419-425, 1999, Characterization of the leukotriene B4 receptor in porcine leukocytes Separation and reconstitution with heterotrimeric GTP-binding proteins). It is known that, in the case of adrenaline receptor, Gs is coupled. According to the report of Loisel et al, Gs from Sf9 cells is expressed and coupled to form a complex on a budded virus (Loisel T P, Ansanay H, St-Onge S, Gay B, Boulanger P, Strosberg A D, Marullo S, Bouvier M., Nat Biotechnol. 1997, November 15(12), pp 1300-1304, Recovery of homogeneous and functional beta 2-adrenergic receptors from extracellular baculovirus particles). Various isoforms of G protein are expressed in Sf9 cells as in mammal cells, but its amounts are different depending on the type of G protein (Leopoldt D, Harteneck C, Numberg R, Naunyn-Schmiedeberg's Arch Pharmacol, 356, pp 216-224, 1997, G Proteins endogenously expressed in Sf9 cells: interactions with mammalian histamine receptors). Since Gs is relatively abundantly present in Sf9 cells, adrenaline receptor expressed in the virus can be couple with Gs from insect cells so that a functional membrane receptor is expressed in the system of Loisel et al. However, in the case of the receptor (for example, leukotriene B4 receptor) which is coupled with other Gα isoform such as Gi which is relatively poor in Sf9 cells, it is difficult to obtain a functional receptor having a high affinity if it is expressed.